In previous studies we have shown that the B. burgdorferi bmp chromosomal gene cluster encoding the paralogous Bmp membrane lipoproteins is present in all B. burgdorferi sensu lato. This cluster forms two complex transcriptional units that are transcribed into variable monocistronic and polycistronic messages to generate different bmp mRNA and gene product concentrations. Nutritional stress in Borrelia burgdorferi triggers the stringent response that modulates expression of many genes and is mediated by the alarmon (p)ppGpp. Our work now indicates that B. burgdorferi has a transcriptionally active rel/Bbu, gene needed for generation of (p)ppGpp and that the presence of (p)ppGpp is associated with the modulation of expression of bmp and other genes under different environmental conditions. The genetic, transcriptional, and biological characteristics of the bmp gene cluster makes it an ideal model to study chromosomal gene regulation both by trans regulators such as (p)ppGpp and sigma factors and by cis DNA sequences. The hypothesis underlying this project is that expression of bmp and other B burgdorferi genes is modulated in trans by the stringent response both directly by (p)ppGpp and indirectly through sigma 54 and sigma S and in cis by the DNA sequences of their 5' upstream promoter regions. The specific aims of this project are: 1) characterize the regulation of rel/Bbu expression and the Rel/Bbu-mediated stringent response on bmp expression during growth of B. burgdorferi in BSKH media, in co-culture with tick cells, and in rat peritoneal culture chambers and ticks; 2) ascertain the roles of sigma S and sigma 54 in modulating bmp expression by the stringent response and their hierarchical interactions in B. burgdorferi in vitro and in vivo using bacterial genetics methods, DNA microarrays and proteomics; and 3) identify putative regulatory proteins binding to 5' upstream DNA sequences of the rpsL, bmpD, bmpC, bmpA and sigma S genes and their DNA binding sites. The proposed experiments will permit us to identify global factors, hierarchical regulatory networks and cis acting DNA sequences involved in the regulation of bmp genes, and will take studies of gene expression and modulation in B. burgdorferi from a descriptive paradigm to a paradigm framed by mechanistic and causal interpretations.